Vinculin is a 120 kDa cytosolic protein that plays a critical role in cell migration and embryogenesis. It is the paradigm of a multifaceted adhesion molecule that is regulated by autoinhibition. We recently determined the crystal structure of the 120 kDa full-length vinculin at atomic resolution. Building upon this structure, we will pursue an in-depth structure-function study in vitro and in cells. The function of vinculin in cells requires binding to a large number of other proteins, but in only a few cases do we have a clear picture of the nature of the interaction. A tight association between vinculin head and tail domains regulates many of these interactions. One likely mechanism of regulation is steric occlusion in the head-tail complex that is relieved upon head-tail dissociation, but there is also good evidence for allosteric regulation. During the first 4-year funding period of this grant, we determined the structure of the vinculin tail and the full-length molecule. We initiated structure-based functional studies of the tail in its interactions with its major ligands - F-actin, acidic phospholipids and paxillin. We will focus our efforts on the interactions between the vinculin tail and F-actin, the tail with phospholipids and paxillin (in collaboration with Dr. Sharon Campbell), and interactions of the vinculin head with its ligands. We will build up pictures of the closed and open forms of the molecule, what conformational changes occur to vinculin domains on binding ligand, and hence the mechanisms of regulated binding. The primary output of this work will be the structural basis of ligand binding and regulation in vitro. This information will then be used to design experiments to test the role of these binding and regulatory functions in cells, which will be done in collaborations with Drs. David Critchley and Susan Craig. [unreadable] [unreadable]